Method of treating multiple sclerosis

ABSTRACT

A method and computer program product for suppressing the symptoms of multiple sclerosis (MS) by irradiating a subject exhibiting the symptoms with a predetermined dose of UV-containing light from a light source and detecting a suppression of the clinical symptoms of MS. In particular, the detected suppression of the clinical symptoms is disassociated from the vitamin D production within the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/308,820, filed on Feb. 5, 2010 and titled “Method ofTreating Multiple Sclerosis,” which is incorporated by reference hereinin its entirety for all purposes.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is a chronic and often debilitating diseaseaffecting approximately 2.5 million people worldwide (Compston A & ColesA, 2002, Multiple sclerosis, Lancet 359(9313):1221-1231). The hallmarkpathological characteristic of MS is the formation of inflammatoryplaques in the central nervous system. The plaques contain a number ofimmune cells which are believed to orchestrate the autoimmune-mediateddestruction of the myelin sheath surrounding neuronal axons (NoseworthyJ H, Lucchinetti C, Rodriguez M, and Weinshenker B G, 2000, Multiplesclerosis. N Engl J Med 343(13):938-952). Demyelination leads to alteredneuronal signal conduction and a myriad of adverse neurologicalsymptoms.

Although the exact cause of MS is unknown, a number of genetic andenvironmental factors are thought to influence MS development (Ebers GC, 2008, Environmental factors and multiple sclerosis. Lancet Neurol7(3):268-277). Epidemiological studies have demonstrated that MSincidence typically follows a latitudinal gradient in both hemispheres.In the northern hemisphere, including Europe and North America, MS ismore common in the northern regions, whereas in the southern hemisphere,including Australia MS is more prevalent in the southern regions (EbersG C and Sadovnick A D, 1993, The geographic distribution of multiplesclerosis: a review. Neuroepidemiology 12(1):1-5). This apparentcorrelation or “latitude gradient” has led to speculation that becausesunlight exposure decreases with increased latitude, decreased sunlightexposure may be an underlying cause of the MS latitude gradient (AchesonE D, Bachrach C A, and Wright F M, 1960, Some comments on therelationship of the distribution of multiple sclerosis to latitude,solar radiation, and other variables. Acta Psychiatr Scand Suppl35(147):132-147). Findings that the average annual hours of sunlightexposure in an individual's place of birth is inversely correlated withMS development support this hypothesis (Acheson E D, Bachrach C A, andWright F M, 1960, Some comments on the relationship of the distributionof multiple sclerosis to latitude, solar radiation, and other variables.Acta Psychiatr Scand Suppl 35(147):132-147; and Sutherland J M, Tyrer JH, and Eadie M J, 1962, The prevalence of multiple sclerosis inAustralia. Brain 85:149-164). Furthermore, individuals with the highestresidential and occupational solar exposure have the lowest rate of MSincidence (Freedman D M, Dosemeci M, and Alavanja M C, 2000, Mortalityfrom multiple sclerosis and exposure to residential and occupationalsolar radiation: a case-control study based on death certificates. OccupEnviron Med 57(6):418-421). These results suggest that decreasedsunlight exposure may be a significant environmental factor contributingto the development of MS.

The sun emits a wide range of electromagnetic radiation, includingultraviolet (UVR) (100-400 nm), visible (400-800 nm), and infrared (800nm) radiation. Exposure to all radiation has profound impacts on humanhealth. For instance, UVR can cause direct damage to DNA and is aleading cause of skin carcinomas. In addition to directly damaging DNA,UVR can induce carcinogenesis by suppressing the immune system (Fisher MS and Kripke M L, 1977, Systemic alteration induced in mice byultraviolet light irradiation and its relationship to ultravioletcarcinogenesis. Proc Natl Acad Sci USA 74(4):1688-1692; and Kripke M L,1974, Antigenicity of murine skin tumors induced by ultraviolet light. JNatl Cancer Inst 53(5):1333-1336).

UVR can also be absorbed by photoreceptors in human cells, resulting inthe release of a number of secondary mediators capable of suppressingcell-mediated immunity through multiple mechanisms (Leitenberger J,Jacobe H T, and Cruz P D, Jr., 2007, Photoimmunology—illuminating theimmune system through photobiology. Semin Immunopathol 29(1):65-70).These mechanisms lead to both local and systemic immunosuppression,thereby eliminating natural defense mechanisms against aberrant cellgrowth.

Although UV-induced immunosuppression clearly has detrimental effects inthe context of skin cancer, it may have beneficial effects onorgan-specific autoimmune diseases such as MS (McMichael A J and Hall AJ, 1997, Does immunosuppressive ultraviolet radiation explain thelatitude gradient for multiple sclerosis? Epidemiology 8(6):642-645). Arecent study demonstrated that MS relapse rates are lower in the summerthan in the winter, suggesting UV exposure may be a contributing factorin relapses (Tremlett H et al., 2008, Monthly ambient sunlight,infections and relapse rates in multiple sclerosis. Neuroepidemiology31(4):271-279). Furthermore, experiments conducted in the experimentalautoimmune encephalomyelitis (EAE) animal model of MS have demonstratedthat seven-day pretreatment with UVR prevents disease induction in SJLmice (Hauser S L et al., 1984, Prevention of experimental allergicencephalomyelitis (EAE) in the SJL/J mouse by whole body ultravioletirradiation. J Immunol 132(3):1276-1281). Our attempt to reproduce thepreventative effect of irradiation discussed in the above-mentionedstudy demonstrated no protective effect (see FIG. 1 and the relateddescription below). Thus, while avoiding UVR exposure may reduce therisk of various skin cancers, it could inadvertently increase the riskof developing autoimmune diseases such as MS.

UVR also modulates the immune response by stimulating the endogenousproduction of vitamin D in the skin. UVB wavelengths between 270 and 300nm stimulate the production of pre-vitamin D₃ from the cholesterolderivative 7-dehydrocholesterol (Jones G, Strugnell S A, and DeLuca H F,1998, Current understanding of the molecular actions of vitamin D.Physiological Reviews 78(4):1193-1231). Pre-vitamin D₃ undergoes aspontaneous isomerization to produce vitamin D₃. Vitamin D₃ undergoestwo successive hydroxylation steps to form the active hormone1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D3). The first activation stepoccurs in the liver, where vitamin D₃ is hydroxylated at carbon-25 togenerate 25-hydroxyvitamin D₃ (25(OH)D₃) (Blunt J W, DeLuca H F, andSchnoes H K, 1968, 25-hydroxycholecalciferol. A biologically activemetabolite of vitamin D3. Biochemistry 7(10):3317-3322). The 25(OH)D₃metabolite is the primary circulating form of vitamin D₃ and is commonlyused as a clinical indicator of vitamin D status (DeLuca H F, 2004,Overview of general physiologic features and functions of vitamin D. AmJ Clin Nutr 80(6 Suppl):1689S-1696S). The second activation step occursin the kidney and involves the stereospecific hydroxylation of 25(OH)D₃at carbon-1 to yield 1,25(OH)₂D₃ (Holick M F, Schnoes H K, and DeLuca HF, 1971, Identification of 1,25-dihydroxycholecalciferol, a form ofvitamin D3 metabolically active in the intestine. Proc Natl Acad Sci USA68(4):803-804; and Fraser D R and Kodicek E, 1970, Unique biosynthesisby kidney of a biological active vitamin D metabolite. Nature228(5273):764-766). The classical biological function of 1,25(OH)₂D3 isto maintain sufficient serum calcium and phosphorus levels for propermineralization of bone and calcium for neuromuscular function.

In addition to its role in regulating serum calcium levels, vitamin Dmay also be an environmental factor in MS and other autoimmune diseases(Hayes C E, Nashold F E, Spach K M, and Pedersen L B, 2003, Theimmunological functions of the vitamin D endocrine system. Cell Mol Biol(Noisy-le-grand) 49(2):277-300). The potential link between vitamin Ddeficit and MS was first proposed by David Goldberg, based on thegeographic “latitude gradient” distribution patterns of MS and therelationship between UVR and vitamin D production (Goldberg P, 1974,Multiple Sclerosis: Vitamin D and calcium as environmental determinantsof prevalence (a viewpoint). Part 1: Sunlight, dietary factors, andepidemiology. International Journal of Environmental Studies 6:19-27).Goldberg postulated that decreased exposure to UVR and subsequentvitamin D insufficiency at higher latitudes pre-disposes individualsresiding in these regions to developing MS.

Much of the evidence supporting this hypothesis is derived fromepidemiological data demonstrating an association between UVR and MSprevalence and the assumption that the immunosuppressive effects of UVRare mediated through vitamin D production. However, UVR suppresses theimmune system through mechanisms independent of vitamin D (Lucas R M andPonsonby A L, 2006, Considering the potential benefits as well asadverse effects of sun exposure: can all the potential benefits beprovided by oral vitamin D supplementation? Prog Biophys Mol Biol92(1):140-149). Therefore, this assumption may not be valid.

Additional evidence suggesting that vitamin D may play a role in MSstems from population-based studies which have correlated high serum25(OH)D₃ levels with a decreased risk for developing MS (Munger K L,Levin L I, Hollis B W, Howard N S, and Ascherio A, 2006, Serum25-hydroxyvitamin D levels and risk of multiple sclerosis. Jama296(23):2832-2838). However, since 25(OH)D₃ levels largely reflect anindividual's exposure to UVR, it is impossible to determine if thedecreased risk is attributable specifically to vitamin D or UVR.

Perhaps the most compelling evidence supporting a role for vitamin D inMS is derived from studies conducted using the experimental autoimmuneencephalomyelitis (EAE) model of MS. A number of in vivo studies havedemonstrated that 1,25(OH)₂D3 can suppress disease induction andprogression in the EAE model of MS (Lemire J M and Archer D C, 1991,1,25-dihydroxyvitamin D3 prevents the in vivo induction of murineexperimental autoimmune encephalomyelitis. J Clin Invest87(3):1103-1107; Cantorna M T, Hayes C E, and DeLuca H F, 1996,1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsingencephalomyelitis, a model of multiple sclerosis. Proc Natl Acad Sci USA93(15):7861-7864; and Nashold F E, Hoag K A, Goverman J, and Hayes C E,2001, Rag-1-dependent cells are necessary for 1,25-dihydroxyvitamin D(3)prevention of experimental autoimmune encephalomyelitis. J Neuroimmunol119(1):16-29). However, complete disease suppression is only achievedusing supraphysiological doses of 1,25(OH)₂D3 which cause vitamin Dtoxicity and hypercalcemia (Cantorna M T, Humpal-Winter J, and DeLuca HF, 1999, Dietary calcium is a major factor in1,25-dihydroxycholecalciferol suppression of experimental autoimmuneencephalomyelitis in mice. Journal of Nutrition 129(11):1966-1971).Vitamin D toxicity and hypercalcemia do not typically occur uponexposure to sunlight due to a number of factors that limit theendogenous production of vitamin D. These factors include thephotochemical conversion of previtamin D₃ into biologically inertcompounds, skin pigmentation, and latitude (Holick M F, MacLaughlin J A,and Doppelt S H, 1981, Regulation of cutaneous previtamin D3photosynthesis in man: skin pigment is not an essential regulator.Science 211(4482):590-593). Thus, the levels of 1,25(OH)₂D₃ required tosuppress MS are well above those that can be produced naturally uponexposure to sunlight. Furthermore, results from our laboratory suggestthat hypercalcemia may be more than simply an unfortunate consequence of1,25(OH)₂D₃ treatment, and may play an essential role in theimmunosuppressive effects of 1,25(OH)₂D₃ (Meehan T F, Vanhooke J, PrahlJ, and Deluca H F, 2005, Hypercalcemia produced by parathyroid hormonesuppresses experimental autoimmune encephalomyelitis in female but notmale mice. Arch Biochem Biophys 442(2):214-221).

Considering the undesired effects that large doses of vitamin D, whichare associated with reduction of clinical parameters of MS, there existsa need for a methodology for suppressing symptoms of MS without thedangerous and unpleasant side effects associated with 1,25(OH)₂D₃treatment.

SUMMARY OF THE INVENTION

Embodiments of the invention address methods and apparatus for treatingand preventing MS that encompass irradiating the subject with a firstdose of light from a light source and detecting a suppression of theclinical symptoms in the subject. According to the embodiments,irradiation is generally characterized by a chosen dose of radiation andrepetition time intervals, and, in a specific embodiment, may becontinuous. In one embodiment, irradiating a subject with light,preferably UV light, is adapted to be unassociated with permanentlyelevated levels of vitamin D and independent from production of vitaminD in the irradiated subject.

In one embodiment, the present invention comprises a method ofsuppressing clinical symptoms of multiple sclerosis comprisingirradiating the subject with a first dose of light from a light sourceand detecting a suppression of the clinical symptoms in this subject.The irradiation may either be with repeated doses or a continuous dose.

One embodiment of the present invention provides a method forsuppressing clinical symptoms of MS in a subject having a referencelevel of serum calcium and a reference level of a serum 25(OH)D₃. Suchmethod includes irradiating the subject with such a first dose of lightfrom a light source that is adapted to cause a change of a serum25(OH)D₃ level in the subject from the reference level of a serum25(OH)D₃ to a first level that is lower than a threshold levelassociated with suppression of the clinical symptoms. In addition, suchmethod may include repeatedly irradiating the subject at repetition timeintervals with a second dose of light from the light source. Here, thesecond dose and repetition time intervals are judiciously chosen as tomaintain a serum 25(OH)D₃ level below the threshold level. Repeatedlyirradiating the subject may require, in one implementation, irradiatingwith the second dose for at least 10 minutes every 24 hours for sevendays. Furthermore, the embodiment includes detecting a suppression ofthe clinical symptoms that is independent of a vitamin D production inthe subject. These repeated doses may be, part of a continuous dose.

In one specific embodiment, the first dose may be further adapted tomaintain the level of serum calcium within 0.5 mg/dL with respect to thereference level of serum calcium, while the second dose and repetitiontime intervals may be further adapted to cause variation of a serum25(OH)D₃ level by no more than 5 ng/mL. In another specific embodiment,each of the first and second doses of light is associated with UVirradiance of at least 2.5 kJ/m² and, alternatively or in addition, withUVB irradiance of at least 2.5 kJ/m².

The suppression of clinical symptoms of MS, according to one embodiment,includes at least one of a decrease of the cumulative disease index(CDI), a delay of onset of MS symptoms, and a reduction of peak ofseverity of MS symptoms, and, in particular, delay or reduction in theappearance of plaques or lesions.

Without the loss of generality, each of the embodiments of the method ofthe invention may further include identifying the subject with the useof pre-defined diagnostic criteria.

Embodiments of the invention further provide a computer program productfor use on a computer system for irradiating a subject, having areference level of serum calcium and a reference level of a serum25(OH)D3, with light from a light source and detecting changes in atleast one of a level of serum calcium and a level of a serum 25(OH)D₃,where the computer program product includes a computer usable tangiblemedium having computer readable program code thereon, and where thecomputer readable program code includes at least a) program code forirradiating the subject with a first dose of light from a light source,the first dose being adapted to cause a change of a serum 25(OH)D₃ levelfrom the reference level of a serum 25(OH)D₃ to a first level that islower than a threshold level associated with suppression of the clinicalparameters; and b) program code for repeatedly irradiating the subject,oriented with respect to a light source, at repetition time intervalswith a second dose of light from the light source, the second dose andrepetition time intervals being such as to maintain a serum 25(OH)D₃level below the threshold level.

In one embodiment, the program code for irradiating the subject with afirst dose includes program code for administering the first doseadapted to maintain the level of serum calcium within 0.5 mg/dL withrespect to the reference level of serum calcium. In addition oralternatively, the program code for repeatedly irradiating the subjectincludes program code for defining such second dose and repetition timeintervals as to not cause variation of a serum 25(OH)D₃ level in excessof 5 ng/mL.

Embodiments of the invention additionally provide a computer programproduct for use on a computer system for irradiating a subject having MSwith light from a light source, where the computer program productincludes a computer usable tangible medium having computer readableprogram code thereon, which, when loaded into the computer system,establishes an apparatus, implemented in the computer system, theapparatus comprising at least an input for receiving a set of energydata characterizing exposure to light prescribed to the subject and aprocessor that operates to determine at least one of components of thelight source and location of said components based on the received setof energy data. In addition, in one embodiment, the apparatus mayinclude an output, in which appears a display of results of theprescribed exposure of the subject to light.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood by referring to thefollowing Detailed Description of Specific Embodiments in conjunctionwith the Drawings, of which:

FIG. 1 shows that UVB pretreatment fails to suppress EAE and causes aslight increase in serum 25(OH)D₃ levels. Mice were treated for 7 daysprior to immunization with the indicated doses of UVB. (A) Averageclinical EAE scores were determined daily for control and UVB treatedmice (n=7-12). (B) Mice were weighed weekly (±SD) throughout the studyto monitor disease-associated weight loss and toxicity. (C) Serumcalcium levels (±SD) were determined at the end of the experiment usinga clinical chemistry analyzer. (D) Serum 25(OH)D₃ levels (±SD) weredetermined at the end of UV treatment and at the termination of theexperiment.

FIG. 2 illustrates that a repeated UVB treatment suppresses EAE andcauses a transient increase in serum 25(OH)D₃ levels. Afterimmunization, mice were treated either every other or every third daywith 2.5 kj/m2 UVB. (A) Average clinical EAE scores were determineddaily for control and UVB treated mice (n=10-11). (B) Mice were weighedweekly (±SD) throughout the study to monitor disease-associated weightloss and toxicity. (C) Serum calcium levels (±SD) were determined at theend of the experiment using a clinical chemistry analyzer. (D) Serum25(OH)D₃ levels (±SD) were determined at selected time point throughoutthe experiment. *P<0.05 compared to control group.

FIG. 3 shows that 25(OH)D₃ only modestly suppresses EAE at doses thatcause severe hypercalcemia. Beginning 10 days prior to immunization,mice were fed a purified 0.87% calcium diet delivering the indicateddoses of either 25(OH)D₃ or 1,25(OH)₂D₃. Treatment continued for theduration of the experiment. (A) Average clinical EAE scores weredetermined daily for vehicle, 25(OH)D₃, and 1,25(OH)₂D₃-treated mice(n=15-17). (B) Mice were weighed weekly (±SD) throughout the study tomonitor weight loss and toxicity. (C) Serum calcium levels (±SD) weredetermined at the end of the experiment using a clinical chemistryanalyzer. (D) Serum 25(OH)D₃ levels (±SD) were determined at thetermination of the experiment. *P<0.05 compared to control group.

DETAILED DESCRIPTION OF THE INVENTION

When used in the specification and in the appended claims, certain termswill have meanings according to the definitions provided below, unlesscontext requires otherwise:

The terms “including” and “comprising” are open-ended terms and shouldbe interpreted to mean “including, but not limited to . . . . ” Theseterms encompass the more restrictive terms “consisting essentially of”and “consisting of.”

The singular forms “a”, “an”, and “the” include plural reference. Aswell, the terms “a” (or “an”), “one or more” and “at least one” can beused interchangeably. The terms “comprising”, “including”,“characterized by” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Each of the publications andpatent documents specifically mentioned herein is incorporated byreference in its entirety for all purposes including describing anddisclosing the chemicals, instruments, statistical analyses andmethodologies which are reported in the publications and which might beused in connection with the invention. All references cited in thisspecification are to be taken as indicative of the level of skill in theart. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Although the exact cause of multiple sclerosis (MS) is unknown, a numberof genetic and environmental factors are thought to influence MSsusceptibility. One potential environmental factor is sunlight and thesubsequent production of vitamin D. A number of studies have correlateddecreased exposure to ultraviolet radiation (UVR) and low serum25-hydroxyvitamin D₃ (25(OH)D₃) levels with an increased risk fordeveloping MS. However, it is unclear whether UVR, vitamin D, or bothare necessary for the putative decrease in MS susceptibility. Theembodiments of the invention, as shown in the Examples below,illustrated the ability of UVR to suppress disease in the EAE model ofMS and allowed to assess the effect of UVR on serum 25(OH)D₃ and calciumlevels. Our results indicated that repeated treatment with UVR, such asdaily treatment, for example, dramatically suppresses clinical signs ofEAE. More importantly, such suppression was associated with only amodest, transient increase in serum 25(OH)D₃ levels which wereinsufficient to suppress EAE independent of UVR treatment. These resultssuggest that UVR is likely suppressing disease independent of vitamin Dproduction and that vitamin D supplementation alone may not replace theability of sunlight to reduce MS susceptibility. Identification of asubject or patient appropriate for treatment of MS symptoms can becarried out based on standardized diagnostic criteria widely used bypracticing physicians, specially in the first stages of the disease,such as the so-called Schumacher and Poser criteria (Compston A, ColesA, October 2008, “Multiple sclerosis”. Lancet 372 (9648): 1502-17;Trojano M, Paolicelli D, November 2001. “The differential diagnosis ofmultiple sclerosis: classification and clinical features of relapsingand progressive neurological syndromes”. Neurol. Sci. 22 (Suppl 2):S98-102; and Poser C M, Brinar V V, June 2004, “Diagnostic criteria formultiple sclerosis: an historical review”. Clin Neurol Neurosurg 106(3): 147-58), or the McDonald criteria, which focus on a demonstrationwith clinical, laboratory and radiologic data of the dissemination of MSlesions in time and space. (Compston A, Coles A, October 2008, “Multiplesclerosis”. Lancet 372 (9648): 1502-17; McDonald W I, Compston A, Edan Get al., July 2001, “Recommended diagnostic criteria for multiplesclerosis: guidelines from the International Panel on the diagnosis ofmultiple sclerosis”. Ann. Neurol. 50 (1): 121-7; and Polman C H,Reingold S C, Edan G et al., December 2005, “Diagnostic criteria formultiple sclerosis: 2005 revisions to the “McDonald Criteria””. Ann.Neurol. 58 (6): 840-6).

The most commonly used diagnostic tools for MS are neuroimaging,analysis of cerebrospinal fluid and evoked potentials. In a positivediagnosis, magnetic resonance imaging (MRI) of the brain and spine showsareas of demyelination (lesions or plaques). Gadolinium administered, asa contrast agent, to a patient with MS typically localizes in these “hotspots” or lesions, and can be easily identified with the use of MRI. TheMRI of the lesions is one of the most efficient methods of diagnosingMS. Measuring the development of new lesions is also a critical andefficient method of monitoring the progression of MS.

MS can be alternatively diagnosed with other known methods. Forinstance, it is known that an MS patient responds less actively tostimulation of the optic nerve (which may be examined using visual andsensory evoked potentials) and sensory nerves due to demyelination ofthese nerve pathways. (Gronseth G S, Ashman E J, May 2000, “Practiceparameter: the usefulness of evoked potentials in identifying clinicallysilent lesions in patients with suspected multiple sclerosis (anevidence-based review): Report of the Quality Standards Subcommittee ofthe American Academy of Neurology”. Neurology 54 (9): 1720-5). Chronicinflammation of the central nervous system can be demonstrated by ananalysis of cerebrospinal fluid. The cerebrospinal fluid is tested foroligoclonal bands, which are present in 75-85% of people with MS.(McDonald W I, Compston A, Edan G, et al., July 2001; and Link H, HuangY M, November 2006, “Oligoclonal bands in multiple sclerosiscerebrospinal fluid: an update on methodology and clinical usefulness”.J. Neuroimmunol. 180 (1-2): 17-28).

The subject chosen for treatment according to an embodiment of theinvention such as, for example, the identified MS patient, can beirradiated or illuminated with light from an appropriate light source.The term “light”, as used herein, encompasses electromagnetic radiationat wavelengths visible to a human eye as well as that within anultraviolet (UV) and near-infrared (near-IR) portions of the spectrum.

The term “light source” generally refers to single or multiplemechanisms or systems serving as a source of illumination inclusive of alight emitter and optical elements that may gate or shape theillumination. Thus, for example, a reflective surface such as a mirrorredirecting at least a portion of light incident upon it, or aphotorefractive element such as a lens, or a spectral filter operatingeither in transmission or reflection that is illuminated with the lightfrom the light emitter is included within the meaning of a “lightsource”. A light source may be used, e.g., for illumination of the MSpatient.

The term “irradiance” is used to describe surface density of lightincident on a reference surface in terms of radiant power per unit areaor, alternatively, in terms of radiant energy per unit area. “Intensity”refers to spatial density of light expressed, for example, as radiantpower per unit solid angle or as radiant energy per unit solid angle.

In one embodiment, a light source may include a light emitter generatinglight, whether at a predetermined wavelength or within at least onespectral band of interest, directly illuminating the patient withintensity and/or irradiance that generally depend on a mutualpositioning of the light source and the patient. For example, andwithout loss of generality, a light emitter such as a fluorescent tube,or a mercury vapor light, or a light-emitting diode (LED), or anincandescent lamp may be used to emit UV light towards the patient.

In the present invention, a preferable light source is chosen to emitlight within the UV-band (e.g., below approximately 400 nm) and, moreparticularly, within the UV-B band, defined as a spectral region betweenapproximately 280 and 315 nm, or in a separate embodiment, within theUV-A band. In another embodiment of the present invention, the spectralregion is between approximately 280 and 290 nm. In an alternativeembodiment of the present invention, the spectral region is between 290and 300 nm. In yet another embodiment of the present invention, thespectral region is between 300 and 315 nm. Various levels ofpatient-exposure to illumination are within the scope of this inventionand, in a specific embodiment, the light source should be configured toassure patient irradiance of at least 2.5 kJ/m². In another embodimentof the invention, the light source also emits non-UV light.

According to embodiments of the invention, the light emitter may besupplemented with auxiliary optical component or a plurality ofcomponents that modifies spatial distribution of light emitted by thelight emitter. For example, the light source may comprise a reflectorintercepting at least a portion of emitted light and redirecting ittowards the subject. Such a reflector may contain a generally curvedreflective surface and, in particular, may incorporate a flat mirror oran optical diffractive element such as a diffractive grating. In aspecific embodiment, a reflector may include a parabolic reflectingsurface that at least partially collimates light emitted by the lightemitter positioned at the focal point of the reflector and redirectsthis light towards the patient that is located at a specified distancefrom the light emitter.

In another embodiment, the light source may contain an optical systemincluding at least one lens that is used to deliver substantiallycollimated light towards the patient. In such a light source, a lightemitter such as a LED may be disposed at or near the focal point of theoptical system. Alternatively, an optical system including at least onelens may be configured to shape the emitted light into a non-collimatedbeam that is further directed towards the subject, which is located atsuch a distance from the light emitter at to assure the exposure of thesubject to the produced illumination at specified levels of irradianceand/or intensity.

In yet another embodiment, the light source may be configured so as toilluminate the subject substantially from all directions. In such anembodiment, the light source may comprise a reflector shaped generallyas a three-dimensional elliptical chamber and substantially surroundingboth the light emitter disposed at or near one focal point of thechamber and the subject located at another focus of the chamber. It isappreciated that, in this case, substantially all of the emitted lightwill be reflected by the internal walls of the chamber towards thesubject.

In a related embodiment of the invention, the light source may includean emitter emitting light within a broad spectral range and at least onespectral filter intercepting the emitted light so as to filter out thelight within a specific spectral band that is preferred for illuminationof the subject. In one implementation where the subject should beilluminated with the UV-light, an optical filter transmitting theUV-light within the specified band (such as UV-B or UV-A) may bedisposed across a collimated beam of light formed by the optical systemof the light source and propagating towards the subject. A variety ofknown optical filters may be used for such purpose such as dichroic andmultichroic filters, interference filters including thin-film filters,for example.

Illumination or irradiation of the subject with light from the lightsource of an embodiment of the invention may be generally carried outwithin a single time period, or repeatedly during severaltime-intervals, or even continuously, as required to achieve aparticular level of light-exposure of the subject.

The overall length of irradiation or treatment is, preferably, definedby a degree of severity of MS exhibited by the patient. In oneembodiment of the present invention, a patient may be exposed to lighttreatment until the most severe of his or her MS symptoms are abated orreduced. In another embodiment of the present invention, the patient maybe exposed to light treatment on a daily basis for as long as relieffrom MS symptoms is desired.

In one embodiment of the present invention, subjects would be irradiateddaily for at least 10 minutes, preferably 10-30 minutes, at a distanceof at least 40 cm from the UV light source. Typically, treatment wouldbe at least 7 days. One may wish to extend treatment either every day orevery other day or every third day for the duration of the treatment. Inanother embodiment, patients may be irradiated with a lower dose oflight but a longer, in some embodiments continuous, interval of lightexposure. For example, one may wish to replace a house-hold light sourcewith a light source capable of emitting a UV light dose suitable for thepresent invention.

One would monitor the patient's MS symptoms and detect a reduction ordelay in these symptoms. Most preferably, the development of new lesionsin the subject would be monitored on a regular (i.e., semi-annual) basisvia MRI as discussed above. Further symptoms that may be monitoredinclude those selected from the group consisting of changes in sensation(hypoesthesia and paraesthesia), muscle weakness, muscle spasms, ordifficulty in moving; difficulties with coordination and balance(ataxia); problems in speech (dysarthria) or swallowing (dysphagia),visual problems (nystagmus, optic neuritis, or diplopia), fatigue, acuteor chronic pain, and bladder and bowel difficulties. Cognitiveimpairment of varying degrees and emotional symptoms of depression orunstable mood are also common. One common clinical measure of disabilityprogression and symptom severity is the Expanded Disability Status Scaleor EDSS.

“Reduction” in MS symptoms is defined to include any significantreduction (at least 30%) of MS symptoms. For instance, in oneembodiment, after six months of daily treatment with the method of thepresent invention one would expect to see at least a 30% reduction inthe amount of new lesions as compared to a MS patient without thetreatment of the present invention.

“Delay” of MS symptoms is defined to include a significant delay (atleast 30%) in the development of MS symptoms. For instance, in oneembodiment, after six months of daily treatment with the method of thepresent invention one would expect to see at least 30% reduction in thesymptoms associated with the lesions on the patients nervous system.With fewer lesions, one would expect less corresponding symptoms,including a delay in, for instance, the appearance of episodic acuteperiods of worsening (i.e. relapses, exacerbation, bouts, attacks, orflare ups). These episodic periods are also susceptible to reduction anddelay and are within the scope of the present invention.

It is appreciated that implementation and/or operation of the embodimentof the invention, as discussed below—including but not limited tooptional calibration and/or tuning of the employed light source,irradiation of the subject under test, detection of changes in clinicalparameters, collection of data representing a process of irradiationand/or detected clinical parameters, and establishing an apparatusimplemented in a computer system—is preferably enabled with the use of aprocessor controlled by instructions stored in a memory. The memory maybe random access memory (RAM), read-only memory (ROM), flash memory orany other memory, or combination thereof, suitable for storing controlsoftware or other instructions and data. Various functions, operations,decisions, etc. of all or a portion of any embodiment of the inventionmay be implemented as computer program instructions, software, hardware,firmware or combinations thereof. Those skilled in the art should alsoreadily appreciate that instructions or programs defining the elementsof an embodiment of the present invention may be delivered to aprocessor in many forms, including, but not limited to, informationpermanently stored on non-writable storage media (e.g. read-only memorydevices within a computer, such as ROM, or devices readable by acomputer 1/O attachment, such as CD-ROM or DVD disks), informationalterably stored on writable storage media (e.g. floppy disks, removableflash memory and hard drives) or information conveyed to a computerthrough communication media, including wired or wireless computernetworks. In addition, while the invention may be embodied in software,the functions necessary to implement the invention may optionally oralternatively be embodied in part or in whole using firmware and/orhardware components, such as combinatorial logic, Application SpecificIntegrated Circuits (ASICs), Field-Programmable Gate Arrays (FPGAs) orother hardware or some combination of hardware, software and/or firmwarecomponents.

Examples of Materials and Methodologies Used for ImplementingEmbodiments of the Invention

Compounds.

25(OH)D₃ and 1,25(OH)₂D₃ were synthesized by Sigma-Aldrich FineChemicals (Madison, Wis.). Compounds were dissolved in absolute ethanol,and the concentration was determined with an ultravioletspectrophotometer using λ_(max) of 264 nm and an extinction coefficientof 18,200 M⁻¹ cm⁻¹ for both compounds. Compounds were added to vegetableoil in the indicated concentrations and delivered in the purified dietas described below.

Animals and Diet.

Female C57BL/6 mice between 7-9 weeks of age were purchased from TheJackson Laboratory (Bar Harbor, Me.). All mice were housed at theUniversity of Wisconsin-Madison Biotron animal facility under specificpathogen-free conditions and exposed to 12 h light-dark cycles. Prior toadministration of experimental diets, mice were fed ad libitum standardrodent Labdiet® 5008 chow (Purina Mills International, Richmond, Ind.).In the indicated experiments, eight week old mice were switched to apurified diet containing all the essential nutrients for normal growth(Smith S M, Levy N S, & Hayes C E, 1987, Impaired immunity in vitaminA-deficient mice. J Nutr 117(5):857-865). 25(OH)D₃ and 1,25(OH)₂D₃ wereadded to the purified diet at doses ranging from 0-1000 μg per kilogrambody weight per day. The diet was delivered in solidified agar formthree times per week beginning 10 days prior to immunization andcontinued until the termination of the experiment. Animal protocols wereapproved by the University of Wisconsin-Madison Institutional AnimalCare and Use Committee.

UV Irradiation.

During preparation, mice from the control and UV-treated groups wereshaved with electric clippers one day before initiating UV-therapy. Inone embodiment, UV-treated mice were irradiated with a bank of fourunfiltered FS20T12 fluorescent sunlamps (Solarc Systems, Barrie, ON)emitting UVR within a broad band of 280-360 nm. Approximately 65% of thelight-output was in the UVB-range (290-320 nm). The radiation output wasmeasured, prior to each treatment, with the use of a UVX radiometerequipped with a 302 nm sensor (UVP, Upland, Calif.). Mice wereindividually irradiated in a 16-chamber plexiglass cage specificallydesigned to prevent mice from shielding each other from the UVR. Becauseof the possibility that the UVB-light output was unequal in thedifferent chambers, mice were rotated through the different chambers onsuccessive days. Mice were irradiated daily for either 13 minutes (2.5kJ/m²) or 26 minutes (5.0 kJ/m²) at a distance of 40 cm from theUV-light source. In the UV-pretreatment study, mice were treated oncedaily with either 2.5 kJ/m² or 5.0 kJ/m² for a total of seven days. Inthe repeated irradiation UV study, mice were treated once daily with 2.5kJ/m² for seven days, then either every other day or every third daywith 2.5 kJ/m² UVB for the duration of the experiment.

Induction of EAE.

Myelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅)(MEVGWYRSPFSRVVHLYRNGK-SEQ ID NO:1) was synthesized at the University ofWisconsin-Madison Biotechnology Center and purified to −95% byreverse-phase HPLC. The MOG₃₅₋₅₅ peptide was resuspended in sterile PBSto a concentration of 4 mg/ml, then emulsified with an equivalent volumeof complete Freund's adjuvant (CFA) supplemented with 5 mg/mlinactivated Mycobacterium tuberculosis H37Ra (DIFCO Laboratories,Detroit, Mich.). EAE was induced in 9-week old C57BL/6 mice bysubcutaneous injection of 100 μl of MOG₃₅₋₅₅/CFA homogenate delivering200 μg of MOG₃₅₋₅₅ peptide. On the day of immunization and 48 h later,mice were injected intraperitoneally with 200 ng of pertussis toxin(List Biological Laboratories, Campbell, Calif.) diluted in sterile PBS.Mice were scored daily for clinical signs of EAE using the followingscale: 0, no clinical disease; 1, loss of tail tone; 2, unsteady gait;3, hind limb paralysis; 4, forelimb paralysis; 5, death. Scoring wasperformed by the same individual throughout the experiment to ensureconsistency. On selected days mice were independently scored by adifferent individual for comparison purposes, but the scores were notcounted in the final analysis.

Analysis of Serum Calcium Levels.

Blood samples were collected at the termination of the experiments andspun at 6000 rpm (2938 g) for 15 min, followed by a second spin at 14000rpm (16883 g) for 1 min. Serum calcium levels were determined using thecalcium L3K reagent (Genzyme Diagnostics, Charlottetown, PE Canada) andthe ABX Pentra 400 clinical chemistry analyzer (Horiba-ABX Diagnostics,Irvine, Calif.).

Analysis of Serum 25(OH)D₃ Levels.

Blood samples were collected at selected time points throughout theexperiment. Red blood cells were removed through two successivecentrifugation steps as described above. Serum 25(OH)D₃ levels weredetermined using a ¹²⁵I-radioimmunological assay following themanufacturer's instructions (DiaSorin, Stillwater, Minn.). Samples abovethe range of the standard curve were diluted prior to analysis.Radioactivity was quantified using a Cobra 5002 gamma scintillationcounter (PerkinElmer, Shelton, Conn.).

Data Analysis.

Individual subjects (mice) were scored daily for signs of EAE, and themean clinical score was calculated for each group. Average onset andseverity were calculated in affected mice displaying a clinical score of≧1.0 for a minimum of two consecutive days. The onset value wascalculated by averaging the first day when clinical signs appeared. Theseverity value was determined by averaging the maximum disease scorereached during the entire experiment. The cumulative disease index (CDI)was calculated by summing the clinical scores for each group for alltime points collected and dividing by the number of mice per group.Statistical analysis was performed using the two-tailed Fisher exactprobability test for incidence rates, the Mann-Whitney non-parametrictest for clinical scores, and the unpaired Student's t test for allother measurements. A value of P<0.05 was considered statisticallysignificant.

EXAMPLES OF EXPERIMENTAL RESULTS

a) UV Pretreatment Slightly Increases 25(OH)D₃ Levels, but does notSuppress EAE.

Hauser et al. (1994) previously reported that the pretreatment with 2.5kJ/m² of UVB light prevented induction of EAE in SJL mice. However,Hauser et al. did not determine the effects on vitamin D production andserum calcium levels. We attempted to confirm the Hauser findings in themyelin oligodendrocyte glycoprotein (MOG) model of EAE and to determinewhat effect UV treatment might have on vitamin D production and serumcalcium levels. In one experiment, we irradiated subjects (femaleC57BL/6 mice) once daily for seven days with UVB-light with irradiancelevels of 2.5 kJ/m² or 5.0 kJ/m². The subjects were immunized withMOG₃₅₋₅₅ following the last UV treatment and monitored daily forclinical signs of EAE. The light sources were appropriately positionedto assure that the mice receive the reported 2.5 kJ/m² used by Hauser etal. In contrast to Hauser et al., treatment with 2.5 kJ/m2 of UVB-lighthad no significant effect on any of the clinical parameters that weretested (see Table 1 and FIG. 1A). Moreover, even the doubled UVBexposure (5.0 kJ/m²) had no significant effect on clinical signs of EAE,although the onset appeared to be slightly delayed.

TABLE 1 UV pretreatment does not suppress clinical signs of EAE. FemaleC57BL/6 mice on a regular chow diet were treated once daily for sevendays with either 2.5 kJ/m² or 5.0 kJ/m² UVB prior to immunization withMOG₃₅₋₅₅. The cumulative disease score (CDI) was calculated by summingall the clinical scores for the entire experiment and dividing by thenumber of mice for each group. The clinical data demonstrate the mean ±SD from one representative of 3 individual experiments. Day of PeakTreatment Incidence Onset Severity CDI Control 100% (7/7) 11 ± 1 3.3 ±0.4 43 ± 7 2.5 kJ/m² 100% (11/11) 12 ± 3 3.3 ± 0.5 42 ± 9 5.0 Kj/m² 92%(11/12) 14 ± 3 3.4 ± 0.6  36 ± 15

Vitamin D toxicity is known to cause weight loss and a dramatic rise inserum calcium levels. To assess the effect of UV treatment on theseparameters, mice were weighed at selected time intervals throughout thestudy, and serum calcium levels were determined at the termination ofthe experiment. As shown in FIG. 1B, UVB-pretreatment did notsignificantly affect the weight of the mice. Furthermore, there were nodetected difference in serum calcium levels at either the end of theUVB-pretreatment period (data not shown) or at the termination of theexperiment (FIG. 1C). In addition, serum 25(OH)D₃ levels were determinedboth at the end of the UVB-pretreatment period and at the termination ofthe experiment. As shown in FIG. 1D, pretreatment with 2.5 kJ/m² and 5.0kJ/m² UVB led to a slight increase, at the end of the UVB-pretreatmentperiod, in serum 25(OH)D₃ levels (75 ng/ml) as compared to the controlgroup (67 ng/ml). Corresponding levels of the 5.0 kJ/m² group remainedelevated at the termination of the experiment. Thus, the conductedUVB-pretreatment did not cause vitamin D toxicity or hypercalcemia, anddid not confer protection against the development or progression of EAE.

b) Repeated Treatment with UV Suppresses Clinical Signs of EAE.

Individuals living in equatorial regions are exposed to UVR on a dailybasis for much of their lives. Although it is not possible to mimic theeffects of a lifetime of UVR exposure in the lab, we reasoned thatperiodic (for example, daily) treatment with UVR throughout theexperiment would provide a more realistic representation of UVR exposurein these regions. To determine the effect of daily UVR-treatment on EAE,mice were treated once daily with 2.5 kJ/m² UVB for seven days prior toimmunization with MOG₃₅₋₅₅. Following the immunization, mice wereadditionally irradiated either every other day or every third day with2.5 kJ/m² UVB-light for the duration of the experiment. As shown inTable 2, the incidence of EAE was not significantly decreased in eithertreatment group as a result of such irradiation. However, treatment with2.5 kJ/m² every third day did cause a slight reduction in diseaseseverity and a decrease in the cumulative disease index (CDI). Asignificant reduction in the average clinical EAE scores was also notedin this group (FIG. 2A). Increasing the frequency of UVB exposure toevery other day enhanced the suppressive effect of the UVB treatment.Treatment with 2.5 kJ/m² UVB every other day significantly delayed theonset of the disease, reduced the peak severity, and decreased the CDIcompared to the control group, as shown in Table 2. By “onset of thedisease” we mean the presence of identifiable lesions in the patient'snervous system as identified via MRI. By “reducing peak severity” wemean reducing the number of new lesions. Increasing the frequency of UVBexposure also caused a further decrease in the average clinical EAEscores (FIG. 2A). Thus, irradiation with UVB-light was far moreeffective at suppressing EAE when treatment was delivered throughout theexperiment, as opposed to the case when such irradiation wasdiscontinued after the immunization of the subjects.

TABLE 2 Repeated UV-treatment inhibits EAE. Female C57BL/6 mice on aregular chow diet were treated once daily for seven days with 2.5 kJ/m²UVB prior to immunization with MOG₃₅₋₅₅. After immunization, mice weretreated either every other or every third day with 2.5 kJ/m² UVB. Theclinical data demonstrate the mean ± SD from one representative of 2individual experiments. Day of Peak Treatment Incidence Onset SeverityCDI Control 100% (11/11) 12 ± 1 3.8 ± 0.7 54 ± 12  2.5 kJ/m² 82% (9/11) 17 ± 3*  2.3 ± 0.9* 17 ± 16* every 2^(nd) day 2.5 kJ/m² 90% (9/10) 14 ±3 3.1 ± 0.9 32 ± 19* every 3^(rd) day *P < 0.05 compared to the controlgroup.

In addition to weight loss caused by vitamin D toxicity, mice can alsolose weight due to muscle wasting and decreased food ingestion secondaryto paralysis during the clinical course of EAE. The loss in weightcorrelated with severity of the disease in mice displaying more severesigns of disease. Mice treated every other day or every third day with2.5 kJ/m² UVB did not lose as much weight as the control group (see FIG.2B). Furthermore, the serum calcium levels in both UVB-treated groupswere normal (FIG. 2C). Serum 25(OH)D₃ levels were significantly elevatedon the day of immunization in both UVB-treated groups (FIG. 2D).However, 25(OH)D₃ levels did not remain elevated despite thecontinuation of UVB treatment. Thus, continuous UVB treatment causedsignificant suppression of clinical signs of EAE without elevating serumcalcium levels and caused only a transient elevation of serum 25(OH)D₃levels.

c) 25(OH)D₃ Fails to Prevent EAE at Doses that Cause SevereHypercalcemia.

After establishing that continuous treatment with UVB suppresses EAEwithout dramatically increasing serum 25(OH)D₃ levels, we sought todetermine if 25(OH)D₃ levels obtained upon UVB treatment were sufficientto suppress EAE without UVB treatment. Female C57BI/6 mice were treatedwith either 10, 500, or 1000 μg/kg 25(OH)D₃ per day and compared to micetreated with vehicle or 2.5 μg/kg 1,25(OH)₂D₃ per day. Pilot studiesindicated that treatment with 2.5 μg/kg of 1,25(OH)₂D₃ per day caused adramatic suppression of clinical signs of EAE and was associated withsevere hypercalcemia (data not shown). Consequently, this dose of1,25(OH)₂D₃ served as a useful treatment group with which to compare theclinical and calcemic effects of 25(OH)D₃.

Treatment with 10 ug/kg of 25(OH)D₃ per day had no significant effect onthe incidence, onset, severity, or progression of EAE (Table 3, FIG.3A). Increasing the dose to 500 μg/kg per day caused a significant delayin the onset of disease and a slight suppression of clinical EAE scores.Further increasing the dose to 1000 μg/kg 25(OH)D₃ per day only slightlyenhanced the suppressive effects seen in the 500 μg/kg 25(OH)D₃treatment group causing a significant decrease in the CDI as well as adelay in the onset of clinical signs of disease compared to the vehiclegroup. Thus, even at a dose as high as 1000 μg/kg per day, 25(OH)D₃caused only a modest suppression of EAE. In contrast, treatment with 2.5μg/kg of 1,25(OH)₂D₃ led to a significant decrease in the diseaseincidence, delayed the onset, and dramatically decreased the CDIcompared to the vehicle and 25(OH)D₃-treated groups.

Treatment with 1000 μg/kg of 25(OH)D₃ and 2.5 μg/kg of 1,25(OH)₂D₃caused a significant decrease in the weight of the mice at thetermination of the study (FIG. 3B). However, the drop in weightdeveloped more slowly and was reduced in magnitude in the 1000 μg/kg25(OH)D₃ group. Serum calcium levels were unchanged in mice treated with10 μg/kg of 25(OH)D₃ (9.9 mg/dl) compared to the vehicle group (10.1mg/dl) (FIG. 3C). In contrast, treatment with 500 μg/kg 25(OH)D₃ (12.9mg/dl), 1000 μg/kg 25(OH)D₃ (14.2 mg/dl), and 2.5 μg/kg 1,25(OH)₂D₃(14.9 mg/dl) all caused hypercalcemia. Although the elevation in serumcalcium levels was similar in the 1000 μg/kg 25(OH)D₃ and 2.5 μg/kg1,25(OH)₂D₃ treated groups, only 1,25(OH)₂D₃ prevented the induction ofEAE (FIG. 3A). Thus, even at doses that dramatically elevated serumcalcium levels and caused weight loss, 25(OH)D₃ provided only modestsuppression of EAE. It is known that at high plasma levels of 25(OH)D₃,it acts as an analog of 1,25(OH)₂D₃ and increases serum calcium levels(Shepard R M & Deluca H F, 1980, Plasma concentrations of vitamin D3 andits metabolites in the rat as influenced by vitamin D3 or25-hydroxyvitamin D3 intakes. Archives of Biochemistry and Biophysics202(1):43-53). Furthermore, this occurs in 1α-hydroxylase null mice(DeLuca, H. F., Prahl, J. and Plum, L. A., in preparation). Although25(OH)D₃ acts as an analog elevating serum calcium levels, it may notexpress all of the functions of 1,25(OH)₂D₃ such as immunomodulation.

TABLE 3 25(OH)D₃ only modestly suppresses EAE. Female C57BL/6 mice weretreated with either 25(OH)D₃ or 1.25(OH)₂D₃ in the indicated dosesdelivered in purified diet. All mice were immunized with MOG₃₅₋₅₅ 10days after initiating therapy with the vitamin D metabolites. Mice weremonitored daily for 25 days and assessed clinically for signs of EAE.The clinical data demonstrate the mean ± SD from one representative of 3individual experiments. Day of Peak Treatment Incidence Onset SeverityCDI Vehicle 87% (13/15) 13 ± 2  2.7 ± 0.8 25 ± 10 10 μg/kg 25 D₃ 88%(15/17) 14 ± 3  2.9 ± 0.9 23 ± 16 500 μg/kg 25 D₃ 82% (14/17) 16 ± 4*2.7 ± 0.6 19 ± 12 1000 μg/kg 25 D₃ 82% (14/17) 16 ± 3* 2.6 ± 0.6  17 ±11* 2.5 μg/kg 1.25 D₃ 35% (6/17)† 20 ± 3† 2.3 ± 0.6  4 ± 7† *P < 0.05compared to the vehicle group. †P < 0.05 compared to all other groups.

Analysis of serum 25(OH)D₃ levels revealed that dietary administrationof 25(OH)D₃ led to a dose-dependent increase of the 25(OH)D₃ metabolitein the serum of treated mice (FIG. 3D). Treatment with 10 μg/kg of25(OH)D₃ resulted in serum 25(OH)D₃ levels similar to those seen uponcontinuous UVB treatment (FIGS. 2D and 3D). Notably, unlike withcontinuous UVB treatment, dietary administration of 10 μg/kg 25(OH)D₃had no effect on EAE progression. This finding suggests that the serum25(OH)D₃ levels obtained upon treatment with UVB are insufficient tosuppress EAE and that UVB likely suppresses EAE independent of vitamin Dproduction.

In contradistinction to Hauser et al., the seven-day pretreatment with2.5 kJ/m² UVB did not suppress clinical signs of EAE. This discrepancyis potentially due to differences in mouse strains and antigens utilizedin these studies or to differences in UV administration. Although UVBpretreatment failed to show an effect on EAE progression, continuous UVBtreatment throughout the duration of the experiment caused,nevertheless, significant inhibition of EAE. This suggests thatincreasing the frequency of UVB exposure enhances its suppressiveeffects, and that the mechanisms underlying disease suppression may betransient and reversible.

Surprisingly, continuous UVB treatment only slightly elevated serum25(OH)D₃ levels. Daily treatment with 2.5 kJ/m² UVB for seven dayscaused a modest increase of 16 ng/ml of 25(OH)D₃ in the serum. However,there was no difference in serum 25(OH)D₃ levels at later time pointsdespite continued exposure to UVB. Further increases in 25(OH)D₃ levelsmay have been inhibited by mechanisms meant to prevent vitamin Dtoxicity. Clinical signs of EAE were observed to remain suppressedthroughout the duration of the study, even when 25(OH)D₃ levels were nolonger elevated compared to control mice. This suggests that sustainedelevations of 25(OH)D₃ levels were not critical for the suppressiveeffects of UVB on EAE. This observation led us to explore the ability of25(OH)D₃ delivered in the diet to suppress EAE independent of UVBexposure. Our results indicate that treatment with 10 μg/kg of 25(OH)D₃had no effect on EAE despite causing an elevation in serum 25(OH)D₃levels similar to that seen in the UVB-treated mice. Furthermore,treatment with up to 1000 μg/kg of 25(OH)D₃ caused only a modestsuppression of EAE and was associated with severe hypercalcemia. Incontrast, continuous treatment with 2.5 kJ/m² UVB led to greater diseasesuppression and had no effect on serum calcium levels. In humans, thenormal range of serum 25(OH)D₃ levels is between 20-100 ng/ml (Holick MF (2009) Vitamin D status: measurement, interpretation, and clinicalapplication. Ann Epidemiol 19(2):73-78). Vitamin D toxicity occurs atserum 25(OH)D₃ levels above 200 ng/ml (Holick M F, 2009). The 25(OH)D₃doses required to suppress EAE were well above this level. Thus, ourdata suggests that the 25(OH)D₃ levels obtained upon treatment with UVBare insufficient to suppress EAE, and that UVB is likely suppressingdisease through mechanisms that are independent of vitamin D production.

The current model used to explain the relationship between increased UVexposure and decreased MS incidence is that UVR is critical forproducing vitamin D which is then converted into 25(OH)D₃. Providedsufficient 25(OH)D₃ levels are present, 25(OH)D₃ can be converted to1,25(OH)₂D₃ and perform immunoregulatory functions that suppressautoimmune mechanisms. Support for this hypothesis is derived fromstudies indicating that decreased exposure to UVR and decreased 25(OH)D₃levels are associated with a higher risk for developing MS (Munger K L,Levin L I, Hollis B W, Howard N S, and Ascherio A, 2006, Serum25-hydroxyvitamin D levels and risk of multiple sclerosis. Jama296(23):2832-2838; and van der Mei I A, et al., 2007, Vitamin D levelsin people with multiple sclerosis and community controls in Tasmania,Australia. J Neurol 254(5):581-590). However, our results suggest thatthe levels of 25(OH)D₃ required to suppress EAE cannot feasibly beproduced upon exposure to UVR.

UVR can suppress the immune system through a number of mechanismsindependent of vitamin D, including inhibiting antigen presentation,altering inflammatory cytokine levels, and inducing suppressor T-cellpopulations (Norval M, McLoone P, Lesiak A, & Narbutt J, 2008, Theeffect of chronic ultraviolet radiation on the human immune system.Photochem Photobiol 84(1):19-28). Therefore, we suggest that UVR islikely playing a role in immunosuppression independent of vitamin Dproduction. Potential caveats to this hypothesis include importantdifferences between the immune systems of mice and humans (Mestas J andHughes C C, 2004, Of mice and not men: differences between mouse andhuman immunology. J Immunol 172(5):2731-2738), as well as between MS andEAE (Steinman L and Zamvil S S, 2005, Virtues and pitfalls of EAE forthe development of therapies for multiple sclerosis. Trends Immunol26(11):565-571). Additionally, the electromagnetic radiation spectrumemitted by UV bulbs is not representative of sunlight and delivers amuch higher proportion of UVB (Brown D B, et al., 2000, Commonfluorescent sunlamps are an inappropriate substitute for sunlight.Photochem Photobiol 72(3):340-344). Despite these potential caveats, ourdata suggests that the putative benefits associated with exposure to UVRcannot be completely recapitulated by simple supplementation withvitamin D. In fact, the benefits of 25(OH)D₃ levels below the thresholdthat causes vitamin D toxicity and hypercalcemia would likely benegligible. Thus, at least some exposure to UVR may be necessary toprevent MS development. More work is required to determine the optimallevels of UVR exposure that provide the beneficial aspects of UVR whileavoiding the detrimental effects associated with chronic UVR exposure.

Additional evidence linking vitamin D and MS is the observation thattreatment with the active form of vitamin D, 1,25(OH)₂D₃, suppresses EAE(23, 24). However, the efficacy of 1,25(OH)₂D₃ treatment is closelylinked with the hormone's ability to increase serum calcium levels;complete disease suppression only occurs using doses of 1,25(OH)₂D₃ thatcause severe hypercalcemia (26). Prolonged hypercalcemia can lead to thecalcification of soft tissues such as kidney, heart, and liver,ultimately leading to organ failure. The hypercalcemic effects of1,25(OH)₂D₃ have precluded its usage as a therapeutic agent in thetreatment MS. A number of investigators have tried to overcome thislimitation by developing less calcemic vitamin D analogs in hopes ofreducing the calcemic effects while retaining the suppressive effects ofthe natural hormone (Lermire J M, Archer D C, and Reddy G S, 1994,1,25-Dihydroxy-24-OXO-16ene-vitamin D3, a renal metabolite of thevitamin D analog 1,25-dihydroxy-16ene-vitamin D3, exertsimmunosuppressive activity equal to its parent without causinghypercalcemia in vivo. Endocrinology 135(6):2818-2821, Mattner F, etal., 2000, Inhibition of Th1 development and treatment ofchronic-relapsing experimental allergic encephalomyelitis by anon-hypercalcemic analogue of 1,25-dihydroxyvitamin D(3). Eur J Immunol30(2):498-508; and van Etten E, et al., 2007, Novel insights in theimmune function of the vitamin D system: synergism with interferon-beta.J Steroid Biochem Mol Biol 103(3-5):546-551). Despite modest success, notreatment involving 1,25(OH)₂D₃ or vitamin D analogs has conclusivelyshown prevention of EAE without elevation of serum calcium levels.Moreover, results from our laboratory suggest that calcium may beplaying an essential mechanistic role in 1,25(OH)₂D₃-mediatedsuppression of EAE (26, 28). These results diminish but do not eliminatethe chance that an analog of 1,25(OH)₂D₃ can be found that may suppressMS. In contrast, continuous treatment with UVB suppresses EAE withoutaltering serum calcium levels. Furthermore, there are no reported casesof hypercalcemia caused by excessive sunlight exposure (30). Thissuggests that disease suppression with UVR is independent of calcium,and that UVR is likely suppressing disease through different mechanismsthan 1,25(OH)₂D₃.

Implementation of embodiments of the invention, as discussed above,suggest that continuous treatment with UVB suppresses clinical signs andsymptoms of EAE. While UVB treatment causes a slight increase in serum25(OH)D₃ levels, this elevation is insufficient to contribute to diseasesuppression. Furthermore, treatment with UVB did not elevate serumcalcium levels, which appears to be a critical step in1,25(OH)₂D₃-mediated suppression of EAE. Therefore, irradiation with UVBis likely to facilitate suppression of EAE independent of vitamin Dproduction. While the invention is described through the above-describedexemplary embodiments, it will be understood by those of ordinary skillin the art that modifications to, and variations of, the illustratedembodiments may be made without departing from the inventive conceptsdisclosed herein. Furthermore, disclosed aspects, or portions of theseaspects, may be combined in ways not listed above. Accordingly, theinvention should not be viewed as being limited to the disclosedembodiment(s).

We claim:
 1. A method for suppressing clinical symptoms of multiplesclerosis (MS) in a subject, the method comprising irradiating thesubject with an effective first dose of light from a light source;wherein the dose is effective to suppress multiple sclerosis symptoms,and detecting a suppression of the clinical symptoms in the subject. 2.A method according to claim 1, further comprising identifying thesubject with the use of pre-defined diagnostic criteria.
 3. A methodaccording to claim 1, wherein the suppression of the clinical symptomsincludes at least one of a delay of onset of MS symptoms, a reduction ofpeak of severity of MS symptoms, and a decrease of the cumulativedisease index (CDI).
 4. A method according to claim 1, wherein themethod additionally comprises a second light dose.
 5. A method accordingto claim 1, wherein the irradiating includes repeatedly irradiating withsuch a dose and at such repetition time intervals as to suppressclinical symptoms and wherein the subject does not develophypercalcemia.
 6. A method according to claim 1, additionally comprisingthe step of irradiating the subject with a second dose of light, whereineach of the first and second doses of light comprises a UV irradiance ofat least 2.5 kJ/m².
 7. A method according to claim 1, wherein thesubject is has a reference level of serum calcium and a reference levelof a serum 25(OH)D3, and wherein the irradiating the subject includes a)irradiating the subject with a first dose of light from a light source,the first dose being adapted to cause a change of a serum 25(OH)D3 levelfrom the reference level of a serum 25(OH)D3 to a first level that islower than a threshold level associated with suppression of the clinicalsymptoms; and b) repeatedly irradiating the subject at repetition timeintervals with a second dose of light from the light source, the seconddose and repetition time intervals being such as to maintain a serum25(OH)D3 level below the threshold level.
 8. A method according to claim7, wherein the detecting includes detecting suppression of the clinicalsymptoms that is independent of the vitamin D production in the subject.9. A method according to claim 7, wherein the first dose is furtheradapted to maintain the level of serum calcium within 0.5 mg/dL withrespect to the reference level of serum calcium.
 10. A method accordingto claim 7, wherein the second dose and repetition time intervals arefurther adapted to cause variation of a serum 25(OH)D₃ level by no morethan 5 ng/mL.
 11. A method according to claim 7, wherein at least one ofthe first and second doses of light comprises a UV irradiance of atleast 2.5 kJ/m².
 12. A method according to claim 7, wherein thesuppression of the clinical symptoms includes delay or reduction in theappearance of plaques or lesions.
 13. A method according to claim 7,wherein the repeatedly irradiating includes irradiating with the seconddose for at least 10 minutes every 24 hours for seven days.
 14. Acomputer program product for use on a computer system for irradiating asubject with light from a light source and detecting changes in clinicalsymptoms of multiple sclerosis (MS) in the subject, the computer programproduct comprising a computer usable tangible medium having computerreadable program code thereon, the computer readable program codeincluding: program code for pre-setting parameters of irradiation withlight from the light source; and program code for operating the lightsource such as to irradiate the subject with a dose of light from thelight source, wherein the dose has pre-set parameters.
 15. A computerprogram product according to claim 14, wherein the program code foroperating the light source includes program code for operating the lightsource that is configured in a predetermined orientation with respect tothe subject being irradiated.
 16. A computer program code according toclaim 14, wherein the program code for pre-setting parameters ofirradiation includes program code for pre-setting repetition timeintervals and a dose of light such as to prevent the subject beingirradiated from developing hypercalcemia.
 17. A computer program codeaccording to claim 16, wherein the program code for pre-settingparameters of irradiation includes program code for pre-settingcontinuous irradiation.
 18. A computer program code according to claim16, wherein the subject has a reference level of serum calcium and areference level of a serum 25(OH)D₃, and wherein the program code foroperating the light source includes a) irradiating the subject with afirst dose of light from the light source, the first dose being adaptedto cause a change of a serum 25(OH)D₃ level from the reference level ofa serum 25(OH)D₃ to a first level that is lower than a threshold levelassociated with suppression of the clinical symptoms; and b) repeatedlyirradiating the subject at repetition time intervals with a second doseof light from the light source, the second dose and repetition timeintervals being such as to maintain a serum 25(OH)D₃ level below thethreshold level.
 19. A computer program code according to claim 18,wherein program code for operating the light source includes programcode for administering the first dose adapted to cause a change of aserum 25(OH)D₃ level from the reference level of a serum 25(OH)D₃ to afirst level, the first level being lower than a threshold levelassociated with suppression of the clinical parameters.
 20. A computerprogram product according to claim 18, wherein the program code foroperating the light source includes program code for administering thefirst dose adapted to maintain the level of serum calcium within 0.5mg/dL with respect to the reference level of serum calcium.
 21. Acomputer program product for use on a computer system for irradiating asubject having multiple sclerosis (MS) with light from a light source,the computer program product comprising a computer usable tangiblemedium having computer readable program code thereon, which, when loadedinto the computer system, establishes an apparatus, implemented in thecomputer system, the apparatus comprising: an input for receiving a setof input data representing levels of irradiation, with the light,prescribed to the subject; a processor determining at least one ofcomponents of the light source and location of said components based onthe received set of energy data; and an output, in which appears adisplay of results of said exposure.